Cloud Point Extraction from Pineapple Juice *

* The experimental work for this chapter was completed during the internship of Boryana Petrova.

The cloud point system, based on ROKAnol NL5, was chosen for the batch extraction from natural fruit juice, due to its lighter micellar phase (see density difference in Figure 6.10). Consequently, less accumulation of solids in the extract was expected. The cloud point extraction with ROKAnol NL5 from pineapple juice was carried out according to the procedure in chapter 5.6., as an ISPR with the internal removal of the extract (see Figure 2.10 a).The steps included defrosting of the feed, mixing of the samples with the surfactant and a final phase separation at 45 °C.

However, it is well known that processing influences the quality and composition of fruit juices [61,78]. Therefore, the influence of the three cloud point extraction steps on the phenolic content in the pineapple juice was preliminary determined.

The evaluation was based on the gallic acid equivalents (GAE) method in chapter 5.11.3. The results for fresh and defrosted juice are presented in Figure 6.17.

Figure 6.17: Phenolic content (GAE) in fresh and defrosted pineapple juice during the stages of the cloud point extraction in laboratory scale. Error bars indicate the standard deviation, N=3.

All process steps influenced the phenolic content of the juice. Initially, the amount of GAE in the fresh and the defrosted juice was above 1200 mg·L^-1. Those values were comparable to reported phenolic content in authentic fruit juice [77].

Freezing the juice to -20 °C led to an only slight reduction of the total phenolic content. That observation was in accordance with the rule, that frosting is the mildest approach for preserving the freshness of plants [61]. However, the

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initial juice juice after mixing juice after heating at 45 °C

phenolic compounds [mg∙L-1] Fresh juice

Defrosted juice

subsequent mixing and heating to 45 °C were characterized by a further reduction of the phenolic concentration in both juices. The observed trend was similar for the mixing step. The defrosted juice was stable during the exposure to 45 °C.

Overall, during the processing, only 44 % and 61% of the GAE remained in the fresh and defrosted feedstock, respectively. As expected, the exposure to air induced the oxidation of the antioxidants and thus decreased the GAE value.

Additionally, probably some of the thermal sensitive compounds were partially degraded at 45 °C [61].

Still, in comparison to processed pineapple drinks from the local market, the concentration of the phenolic compounds was 100-times higher. Hence, the cloud point extraction of the pineapple juice was beneficial at the proposed conditions.

Whereby, the defrosted juice was more suitable as feedstock. Therefore, a cloud point extraction with ROKAnol NL5 was investigated.

The process performance was evaluated by their phenolic content. The GAE was determined in the feed after the mixing. Subsequently, the phenolic contents in the micellar and the aqueous phases were compared to the initial sample.

Additionally, the antioxidant capacity was determined using the DPPH reduction according to the method in 5.11.4. The corresponding results are depicted in Figure 6.18.

Figure 6.18: Phenolic content (GAE) and antioxidant capacity of the in the feed compared to the micellar and to aqueous phases after the cloud point extraction with ROKAnol NL5 at 45 °C. Error bars indicate the standard deviation, N=3.

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The initial GAE concentration in the feed was lower than the phenolic content in the micellar phase. Additionally, as depicted in Figure 6.18, the GAE fraction in the aqueous phase was comparable to the initial value. Thus, an accumulation of phenolic compounds in the surfactant-rich phase was achieved during the CPE with ROKAnol NL5. Additionally, the reduction of the phenolic content in the aqueous phase was similar to the observations for defrosted juice (see figure Figure 6.17).

That proved the concept that despite the reduction of the GAE value in the juice an accumulation of phenolic compounds using the cloud point extraction is possible. However, the yield of phenolic compounds in the extract was equal to 27

%. In comparison to the batch cloud point extraction of the model solute cinnamic acid (YBatch= 69 %), the observed yield from the authentic feedstock was lower.

On the one hand, the degradation of the phenolic compounds may be responsible for the limited accumulation. On the other hand, the phenolic compounds in the pineapple juice might have lower affinity to solubilize in the micelles than the cinnamic acid. The incomplete recovery of phenolic compounds in a single-stage extraction was in the range of to the yields, observed by Gortzi et al. The authors reported yields of approx. 30 % for the model phenolic compounds gallic acid, rutin and epicatechin in the cloud point system of Genapol X-080 [111]. The recovery of phenolic compounds from 17 – 36 % was also reported for the extraction from fresh apple, sweet lemon and mango juices using different nonionic surfactants [115]. The limited yield of phenolic compounds could be enhanced by performing the cloud point extraction in continuous mode. However, the limited amount of feedstock was not sufficient to perform such experiments in technical scale.

Nevertheless, according to the mass balance, 90 % of the initial GAE were distributed between the coexisting phases. The value was higher than the results obtained for surfactant-free juice. The reason for that may be the preserving effect of ROKAnol NL5 micelles against interaction of the phenolic compounds with the environment.

Gallic acid and its equivalent molecules possess a high antioxidant activity. The reduction of the DPPH, presented in Figure 6.18, corresponded to the antioxidative capacity before and after the cloud point extraction. The amount reduced DPPH per gram sample was similar for the samples of the feed and the extract phase.

Hence, the antioxidative effect was preserved in the micellar phase. However, a reduction of the DPPH-value was obtained for the aqueous phase. That was

contrary to the GAE-value in the surfactant-lean phase. It could be assumed, that the decrease in the antioxidant capacity was due to reduction reactions induced by air exposure and heating. Therefore, the UV-signal at 275 nm was unchanged since the aromatic group was still present in the solution. On the other hand, there were no available groups for the DPPH reduction. In can be concluded, that the observations regarding the antioxidant capacity were further evidence for the preserving effect of the surfactant in the micellar phase. The same tendency was reported for the antiradical activity of the micellar extract from the red-flesh orange juice. However, the authors did not present a more in-depth study on the influence of the micelles on the antioxidant activity [7].

Pineapple juice contains a significant amount of sugars. Solutes such as glucose and fructose can influence the phase separation in cloud point systems [90].

Therefore, the distribution of reducing sugars between the coexisting phases was analyzed according to the method in chapter 5.11.5. The comparison between the sugar content in the initial mixture to the micellar and the aqueous phase is presented in Figure 6.19.

Figure 6.19: Sugar content in the feed compared to the micellar and to the aqueous phases after the cloud point extraction with ROKAnol NL5 at 45 °C. Error bars indicate the standard deviation, N=3.

The concentration of reducing sugars in the feed was 38 g·L^-1. Subsequently, an uneven sugar distribution was observed. A two-times higher sugar concentration was constituted in the aqueous phase in comparison to the micellar phase. That result was in agreement with the observations by Ritter et al. In their contribution, the authors implemented sugars to elevate the density difference between the lighter micellar and the aqueous phase. Hence, it was possible to stabilize the cloud

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point system for the continuous extraction in a stirred column. That was a result of the accumulation of the hydrophilic sugars in the surfactant-lean phase [90].

The same effect can be expected in case of the CPE from fruit juices. The influence of the fruit sugars can lead to more stable phase separation in a biphasic system with lighter micellar phase. Therefore, the sugar content must be considered when designing a direct product recovery from natural feedstock using a surfactant solution.

Lastly, a yellow coloring was observed in the micellar phase. The primary extract in depicted in the most left of Figure 6.20.

Figure 6.20: Pigment accumulation in the micellar phase. From left to right: no dilution, 1:2, 1:3, 1:5 and 1:10 dilutions.

The observed yellow color was evenly distributed and stable at further dilution, as illustrated in Figure 6.20. Additionally, the texture was homogeneous and free of visible solids. The coloring of the micellar phase was due to the accumulation of pigments. The exhibited affinity of the micellar phase to accumulate tocopherol, carotenoids or other yellow and red pigments was reported previously [6,7,122].

Such solubilized colorants can evenly distribute in beverages with high water content, without changing the texture [92]. Hence, the micellar phase could be applied as coloring agent with antioxidant activity in cosmetic formulations.

Overall, the cloud point extraction of phenolic compounds with the cosmetic-grade surfactant ROKAnol NL5 was feasible in batch mode. The conditions used for the separation of the model solute were applicable with the fruit juice as well. After maintaining stable phase separation, an accumulation of phenolic compounds and colorant was achieved. The target compounds maintained their antioxidant capacity into the micellar phase. Moreover, the sugars in the feed lead to further stabilization of the phase separation. Finally, a micellar phase with homogeneous

texture was produced. It can be concluded, the cloud point extraction with ROKAnol NL5 is suitable for the isolation of valuable compounds from fruit juices.

In addition, the extract phase can be utilized as an ingredient for cosmetic formulations, due to the permission of the surfactant in such products. Ultimately, the concept for a cloud point extraction from a natural feedstock using leave-in surfactants was successfully implemented with the pineapple juice.

The results confirmed the suitability of the cloud point extraction for the recovery of valuable compounds from the sensitive fruit juice. In addition, the mild technique is also attractive for the product removal from living cell suspensions, such as green microalgae. The feasibility of the continuous cloud point extraction from microalgal cultures is thus presented in the next chapter.